Anti-viral compounds

ABSTRACT

The present invention provides compounds which inhibit. an envelope virus by inhibiting the fusion of the virus with the host cell. The virus may be inhibited in an infected cell, a cell susceptible of infection or a mammal in need thereof.

CROSS-REFERENCE

This application is a 371 of PCT/US97/07526 filed on May 2, 1997 nowWO97/41860 Nov. 13, 1997 which claims the benefit of U.S. ProvisionalPatent Application Ser. No. 60/016,926 filed on May 6, 1996.

Influenza viruses cause an infectious disease for which there is noadequate therapeutic agent. The disadvantages of existing treatmentsinclude the onset of clinical resistance within thirty six hours and theineffectiveness of the agents against influenza B. Killed influenzavirus vaccines have been available for over sixty years. However, thesevaccines have not lessened the morbidity, mortality or severe financialloss caused by this disease. It follows that an agent which treats orprevents an influenza infection or is effective at preventing theclinical symptoms associated with an influenza infection will result ina significant benefit to society.

Currently, the only compounds approved for the therapeutic andprophylactic treatment of influenza infections are the adamantanes:amantadine and rimantadine. These compounds inhibit influenza A byinhibiting the function of the M2 ion channel activity of the virus.Amantadine is a potent in vitro inhibitor of influenza A virus asdemonstrated by standard antiviral assays such as the plaque reductionassay. Amantadine is effective in reducing the duration of fever andother systemic complaints including but not limited to myalgia (muscularache) and fatigue when administered to individuals infected withinfluenza A within forty-eight hours of the onset of clinical symptoms.It has also been observed that amantadine results in a one hundred-folddecrease of virus titer in the nasal washes of human volunteers infectedwith wild-type influenza virus which correlates with a dramatic decreasein fever score. Thus, in vitro influenza inhibition is predictive ofuseful in vivo effects, i.e. a reduction of the clinical symptomsassociated with the influenza infection.

The present invention derives from the fact that influenza is anenveloped virus which dictates that the virus envelope must be fusedwith the endosomal membrane of the host cell in order to initiate theprocess of introducing its genetic information into the cell. Becausethis process is common to all enveloped viruses, it is an attractivetarget for antiviral chemotherapy. Examples of envelope viruses whichare inhibited according to the present invention include influenza,bovine diarrheal, hepatitis C, tick borne encephalitis and the like. Thefusion domain of the envelope glycoprotein of influenza, hemagglutinin(HA) has been well-characterized. See, White J. M., Annu. Rev. Physiol.vol. 52, pages 675-697 (1990) which is herein incorporated by reference.

Influenza virus HA provides at least two distinct functions: 1)recognition of the host cell receptor, i.e., sialic acid residues onglycoconjugates, and 2) fusion of the viral envelope with the endosomalmembrane. Both functions are essential for the propagation of influenzavirus in vitro and in vivo. During viral maturation, monomeric HA isinserted into a lipid bilayer, post-translationally modified andoligomerized into a trimer of identical subunits (trimeric HA). Theinfectivity of the progeny virus is contingent upon a site-specificcleavage of HA by host cell protease(s). This cleavage results in theformation of two polypeptide chains, HA1 and HA2, which remainassociated by non-covalent interactions as well as by an intermolecularand intramolecular disulfide bonds.

It has been established that influenza HA has two functionally relevantconformations. One conformation (Form A) exists as a metastablestructure at neutral pH and mediates receptor recognition. Followingreceptor mediated binding to the host cell, the virus is transported tothe endosomal compartment where it encounters an acidic environment. Thelow pH triggers a dramatic structural rearrangement of HA (Form A) whichresults in the formation of the other, more stable conformation of HA(Form B).

Form B of HA is required for fusion of the virus envelope with theendosomal membrane. It is the structural rearrangement from Form A toForm B of HA that allows the fusion domain of HA to directly interactwith the endosomal membrane enabling the release of viral geneticinformation into the host cell cytoplasm. These considerations lendthemselves to the development of a strategy for antiviral interventionbased on the abrogation of HA-mediated fusion of virus-host membranes.

The present invention relates to a compound of the formula: ##STR1##wherein: R⁰ and R¹ are independently hydrogen, hydroxy, C₁ -C₆ alkyl, C₁-C₆ alkoxy, hydroxy(C₁ -C₆ alkyl), sulfhydryl, sulfamyl, --SO₂ --Cl,--S--C(O)--N(CH₃)₂, amino, C₁ -C₄ alkylamino, di(C₁ -C₄ alkyl)amino, C₁-C₄ alkylsulfonylamino, di(C₁ -C₄ alkylsulfonyl)amino --X⁰ --O--C(O)--C₁-C₄ alkyl, --O--(X¹)_(i) --X², --C(O)--X³, --N--C(O)--R² or --O--R³ ;

X⁰ is a bond or divalent(C₁ -C₆ alkyl);

X¹ is an amino acid;

X² is hydrogen or an amino protecting group;

i is 1, 2 or 3;

X³ is C₁ -C₆ alkyl, C₁ -C₆ alkoxy, halo(C₁ -C₆ alkyl), hydroxy(C₁ -C₆alkyl) or phenyl;

R² is C₁ -C₄ alkyl, C₁ -C₄ alkoxy, halo(C₁ -C₄ alkyl), hydroxy(C₁ -C₄alkyl), phenyl, p-methoxy-phenyl, p-fluoro-phenyl, naphthyl, pyridyl,piperidinyl, thiazolyl, oxazolyl, thienyl, furyl, tetrahydrofuryl orcyclohexyl;

R³ is C₀ -C₆ alkenyl, --CH₂ --R^(3a), --C(O)--R^(3b), --C(S)--R^(3c),--C(CH₃)₂ C(O)NH₂, phenyl or a group of the formula: ##STR2## R^(3a) isphenyl, p-fluorophenyl, pyridyl, pyrrolidinyl, piperidinyl, piperazinyl,morpholinyl, N--(C₁ -C₄ alkoxycarbonyl)piperidinyl,N-(trifluoromethyl)-piperidinyl, thiazolyl, oxazolyl, imidazolyl,isothiazolyl, isooxazolyl, quinolyl, isoquinolyl, thienyl, furyl,tetrahydrothienyl, tetrahydrofuryl, cyclohexyl, cyclopentyl, cyclopropylor naphthyl;

R^(3b) is pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, N--(C₁-C₄ alkoxycarbonyl)piperidinyl, N--(trifluoromethyl)piperidinyl,benzyloxy, pyridylmethyloxy, C₁ -C₆ alkoxy, halo(C₁ -C₄ alkoxy), amino,C₁ -C₄ alkylamino or di(C₁ -C₄ alkyl)amino;

R^(3c) is amino, C₁ -C₄ alkylamino or di(C₁ --C₄ alkyl)amino;

R^(3d) is oxygen, hydroximino, hydrazino or ═CHZ;

Z is hydrogen, C₁ -C₄ alkyl, halogen, di(C₁ --C₄ alkyl)amino, C₁ -C₄alkoxycarbonyl, carbamoyl(C₁ -₄ alkyl), N--(C₁ -C₄ alkyl)carbamoyl orN,N-di(C₁ -C₄ alkyl)carbamoyl;

R^(3e) is hydrogen, nitro or trifluoromethyl;

X is a bond or --(CH₂)--;

R⁴ is hydrogen, hydroxy, amino, C₁ -C₄ alkylamino, di(C₁ --C₄alkyl)amino, C₁ -C₄ alkoxy, ═O, --O--S(CH₃)₂ C(CH₃)₃, C₂ -C₆alkanoyloxy, N--(C₂ -C₆ alkanoyl)amino, ═N--R⁵ or R⁴ and R⁶ combine toform a bond;

R⁵ is hydroxy, amino, C₁ -C₄ alkylamino, di(C₁ -C₄ alkyl)amino, C₁ -C₄alkoxy, pyridylmethoxy, benzyloxy, piperazinyl, N-(methyl)piperazinyl or--O--CH₂ --C(O)--R^(5a) ;

R^(5a) is hydroxy or C₁ -C₄ alkoxy;

R⁶ is hydrogen, halo, C₁ -C₄ alkyl or ═O;

R⁷ is hydrogen or C₁ -C₄ alkyl;

R⁸ is hydroxy, halo, C₁ -C₆ alkoxy, pyrrolidinyl, piperidinyl,piperazinyl, 4-methyl-piperazinyl, morpholinyl or --N(R⁹)--R¹⁰ ;

R⁹ is hydrogen or methyl;

R¹⁰ is -(divalent C₁ -C₆ alkyl)--R^(10a) ;

R^(10a) is pyridyl,

with the proviso that

i) when R⁴ is hydrogen or ═O, then R⁰ and R¹ cannot both be hydrogen;

ii) when R⁰ cannot be isopropyl; and R⁴ is hydrogen; and R¹ cannot behydrogen, hydroxy or methoxy;

iii) when R⁰ is hydrogen; and R⁴ is hydrogen; then R¹ cannot behydrogen, hydroxy, methoxy, --C(O)CH₃ or --OC(O)CH₃ ; or apharmaceutically acceptable salt thereof.

The present invention provides new compounds of formula I, as describedabove, that are useful for treating or preventing a viral infectionwhere the virus is an envelope virus that undergoeshemagglutinin-mediated fusion with a host cell and/or the resultantsymptoms. These compounds, their pharmaceutically acceptable salts andthe corresponding pharmaceutical formulations can be used alone or incombination with other antivirals, immunomodulators, antibiotics orvaccines.

All temperatures stated herein are in degrees Celsius (° C.). All unitsof measurement employed herein are in weight units except for liquidswhich are in volume units.

The term "halo" represents chloro, fluoro, bromo or iodo.

The term "C₁ -C₆ alkyl" represents a straight or branched alkyl chainhaving from one to six carbon atoms. Typical C₁ -C₆ alkyl groups includemethyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyland the like. The term "C₁ -C₆ alkyl" includes within its definition theterm "C₁ -C₄ alkyl."

The term "halo(C₁ -C₆)alkyl" represents a straight or branched alkylchain having from one to six carbon atoms with 1, 2 or 3 halogen atomsattached to it. Typical halo(C₁ -C₆)alkyl groups include chloromethyl,2-bromoethyl, 1-chloroisopropyl, 3-fluoropropyl, 2,3-dibromobutyl,3-chloroisobutyl, iodo-t-butyl, trifluoromethyl and the like.

The term "hydroxy(C₁ -C₆)alkyl" represents a straight or branched alkylchain having from one to six carbon atoms with an hydroxy group attachedto it. Typical hydroxy(C₁ -C₆)alkyl groups include hydroxymethyl,2-hydroxyethyl, 1-hydroxyisopropyl, 2-hydroxypropyl, 2-hydroxybutyl,3-hydroxyisobutyl, hydroxy-t-butyl, hydroxypentyl and the like.

The term "C₁ -C₄ alkylamino" represents a straight or branchedalkylamino chain having from one to four carbon atoms attached to anamino group. Typical C₁ -C₄ alkylamino groups include methylamino,ethylamino, propylamino, isopropylamino, butylamino, sec-butylamino andthe like.

The term "di(C₁ -C₄)alkylamino" represents a straight or brancheddialkylamino chain having two alkyl chains, each having independentlyfrom one to four carbon atoms attached to a common amino group. Typicaldi(C₁ -C₄)alkylamino groups include dimethylamino, ethylmethylamino,methylisopropyl-amino, t-butylisopropylamino, di-t-butylamino and thelike.

The term "C₁ -C₆ alkoxy" represents a straight or branched alkyl chainhaving from one to six carbon atoms attached to an oxygen atom. TypicalC₁ -C₆ alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy,butoxy, t-butoxy, pentoxy and the like. The term "C₁ -C₆ alkoxy"includes within its definition the term "C₁ -C₄ alkoxy".

The term "C₂ -C₆ alkeny", represents a straight or branched alkenylchain having from two to six carbon atoms. Typical C₂ -C₆ alkenyl groupsinclude ethenyl, propenyl, isopropenyl, buten-2-yl, t-butenyl,penten-1-yl, hexen-3-yl, 3-methylpentenyl and the like.

The term "C₁ -C₄ alkoxycarbonyl" represents a straight or branchedalkoxy chain having from one to four carbon atoms attached to a carbonylmoiety. Typical C₁ -C₄ alkoxycarbonyl groups include methoxycarbonyl,ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl,t-butoxycarbonyl and the like.

The term "carbamoyl(C₁ -C₄)alkyl" represents a straight or branchedalkyl chain having from one to four carbon atoms with a carbamoyl groupattached to it. Typical carbamoyl(C₁ -C₄)alkyl groups includecarbamoylmethyl, carbamoylethyl, carbamoylpropyl, carbamoylisopropyl,carbamoylbutyl and carbamoyl-t-butyl and the like.

The term "N-(C₁ -C₄)alkylcarbamoyl" represents a straight or branchedalkyl chain having from one to four carbon atoms attached to thenitrogen atom of a carbamoyl moiety. Typical N-(C₁ -C₄ alkyl)carbamoylgroups include N-methylcarbamoyl, N-ethylcarbamoyl, N-propylcarbamoyl,N-isopropylcarbamoyl, N-butylcarbamoyl, N-t-butylcarbamoyl and the like.

The term "N,N-di(C₁ -C₄ alkyl)carbamoyl" represents a straight orbranched alkyl chain having a straight or branched C₁ -C₄ alkyl chainattached to each of the nitrogen atoms on a carbamoyl moiety. TypicalN-(C₁ -C₄)alkylcarbamoyl groups include N,N-dimethylcarbamoyl,N-ethyl-N-methylcarbamoyl, N-propyl-N-butylcarbamoyl,N,N-diisopropylcarbamoyl, N-methyl-N-butylcarbamoyl and the like.

The term "C₁ -C₄ alkylsulfonylamino" represents a straight or branchedalkyl group having from one to four carbon atoms attached to asulfonylamino moiety. Typical C₁ -C₄ alkylsulfonylamino groups includemethylsulfonylamino, ethylsulfonylamino, propylsulfonylamino,isopropylsulfonylamino, butylsulfonylamino, isobutylsulfonylamino,sec-butylsulfonylamino and t-butylsulfonylamino.

The term "di(C₁ -C₄ alkylsulfonyl)amino" represents two C₁ -C₄alkylsulfonyl moieties attached to an amino moiety. Typical di(C₁ -C₄alkylsulfonyl)amino groups include methylmethylsulfonylamino,ethylmethylsulfonylamino, propylethylsulfonylamino,isopropylmethylsulfonylamino, t-butylethylsulfonylamino,butylbutylsulfonylamino and the like.

The term "C₂ -C₆ alkanoyl" represents a straight or branched alkyl chainhaving from one to five carbon atoms attached to a carbonyl moiety.Typical C₂ -C₆ alkanoyl groups include ethanoyl, propanoyl,isopropanoyl, butanoyl, t-butanoyl, pentanoyl, hexanoyl,3-methylpentanoyl and the like.

The term "C₂ -C₆ alkanoyloxy" represents a straight or branched alkylgroup having from one to five carbon atoms attached to a carbonyloxymoiety. Typical C₂ -C₆ alkanoyloxy groups include ethanoyloxy,propanoyloxy, isopropanoyloxy, butanoyloxy, isobutanoyloxy,sec-butanoyloxy, t-butanoyloxy, pentanoyloxy and the like.

The term "C₂ -C₆ alkanoylamino" represents a straight or branched alkylgroup one to five carbon atoms attached to a carbonylamino moiety.Typical C₂ -C₆ alkanoylamino groups include ethanoylamino,propanoylamino, isopropanoylamino, butanoyl-amino, isobutanoylamino,sec-butanoylamino, t-butanoylamino, pentanoylamino and the like.

As mentioned above, the invention includes the pharmaceuticallyacceptable salts of the compounds defined by formula I. Althoughgenerally neutral, a compound of this invention can possess asufficiently acidic, a sufficiently basic, or both functional groups,and accordingly react with any of a number of inorganic bases, andinorganic and organic acids, to form a pharmaceutically acceptable salt.

The term "pharmaceutically acceptable salt" as used herein, refers tosalts of the compounds of the above formula which are substantiallynon-toxic to living organisms. Typical pharmaceutically acceptable saltsinclude those salts prepared by reaction of the compounds of the presentinvention with a mineral or organic acid or an inorganic base. Suchsalts are known as acid addition and base addition salts.

Acids commonly employed to form acid addition salts are inorganic acidssuch as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuricacid, phosphoric acid and the like, and organic acids such asp-toluenesulfonic, methanesulfonic acid, oxalic acid,p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid,benzoic acid, acetic acid, and the like.

Examples of such pharmaceutically acceptable salts are the sulfate,pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutyrate, caproate,heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate,phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,γ-hydroxybutyrate, glycollate, tartrate, methanesulfonate,propanesulfonate, naphthalene-1-sulfonate, napththalene-2-sulfonate,mandelate and the like. Preferred pharmaceutically acceptable acidaddition salts are those formed with mineral acids such as hydrochloricacid and hydrobromic acid, and those formed with organic acids such asmaleic acid and methanesulfonic acid.

Base addition salts include those derived from inorganic bases, such asammonium or alkali or alkaline earth metal hydroxides, carbonates,bicarbonates, and the like. Such bases useful in preparing the salts ofthis invention thus include sodium hydroxide, potassium hydroxide,ammonium hydroxide, potassium carbonate, sodium carbonate, sodiumbicarbonate, potassium bicarbonate, calcium hydroxide, calciumcarbonate, and the like. The potassium and sodium salt forms areparticularly preferred.

It should be recognized that the particular counterion forming a part ofany salt of this invention is not of a critical nature, so long as thesalt as a whole is pharmacologically acceptable and as long as thecounterion does not contribute undesired qualities to the salt as awhole.

The term "amino-protecting group" as used in the specification refers tosubstituents of the amino group commonly employed to block or protectthe amino functionality while reacting other functional groups on thecompound. Examples of such amino-protecting groups include formyl,trityl, phthalimido, trichloroacetyl, chloroacetyl, bromoacetyl,iodoacetyl groups, or urethane-type blocking groups such asbenzyloxycarbonyl, 4-phenylbenzyloxycarbonyl, 2-methylbenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, 4-fluorobenzyloxycarbonyl,4-chlorobenzyloxycarbonyl, 3-chlorobenzyloxycarbonyl,2-chlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl,4-bromobenzyloxycarbonyl, 3-bromobenzyloxycarbonyl,4-nitrobenzyloxycarbonyl, 4-cyanobenzyloxycarbonyl, t-butoxycarbonyl,2-(4-xenyl)isopropoxycarbonyl, 1,1-diphenyleth-1-yloxycarbonyl,1,1-diphenylprop-1-yloxycarbonyl, 2-phenylprop-2-yloxycarbonyl,2-(p-toluyl)-prop-2-yloxycarbonyl, cyclopentanyloxycarbonyl,1-methylcyclopentanyloxycarbonyl, cyclohexanyloxycarbonyl,1-methylcyclohexanyloxycarbonyl, 2-methylcyclohexanyloxycarbonyl,2-(4-toluylsulfonyl)-ethoxycarbonyl, 2-(methylsulfonyl)ethoxycarbonyl,2-(triphenylphosphino)-ethoxycarbonyl, fluorenylmethoxycarbonyl("FMOC"), 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl,1-(trimethylsilylmethyl)prop-1-enyloxycarbonyl,5-benzisoxalylmethoxycarbonyl, 4-acetoxybenzyloxycarbonyl,2,2,2-trichloroethoxycarbonyl, 2-ethynyl-2-propoxycarbonyl,cyclopropylmethoxycarbonyl, 4-(decyloxy)benzyloxycarbonyl,isobornyloxycarbonyl, 1-piperidyloxycarbonyl and the like;benzoylmethylsulfonyl, 2-nitrophenylsulfenyl, diphenylphosphine oxideand like amino-protecting groups. The species of amino-protecting groupemployed is not critical so long as the derivatized amino group isstable to the condition of subsequent reaction(s) on other positions ofthe intermediate molecule and can be selectively removed at theappropriate point without disrupting the remainder of the moleculeincluding any other amino-protecting group(s). Preferredamino-protecting groups are t-butoxycarbonyl (t-Boc), allyloxycarbonyland benzyloxycarbonyl (CbZ). Further examples of groups referred to bythe above terms are described by J. W. Barton, "Protective Groups inOrganic Chemistry", J. G. W. McOmie, Ed., Plenum Press, New York, N.Y.,1973, Chapter 2, and T. W. Greene, "Protective Groups in OrganicSynthesis", John Wiley and sons, New York, N.Y., 1981, Chapter 7.

The term "carboxy-protecting group" as used in the specification refersto substituents of the carboxy group commonly employed to block orprotect the carboxy functionality while reacting other functional groupson the compound. Examples of such carboxy-protecting groups includemethyl, p-nitrobenzyl, p-methylbenzyl, p-methoxybenzyl,3,4-dimethoxybenzyl, 2,4-dimethoxybenzyl, 2,4,6-trimethoxybenzyl,2,4,6-trimethylbenzyl, pentamethylbenzyl, 3,4-methylenedioxybenzyl,benzhydryl, 4,4'-dimethoxybenzhydryl, 2,2',4,4'-tetramethoxybenzhydryl,t-butyl, t-amyl, trityl, 4-methoxytrityl, 4,4'-dimethoxytrityl,4,4',4"-trimethoxytrityl, 2-phenylprop-2-yl, trimethylsilyl,t-butyldimethylsilyl, phenacyl, 2,2,2-trichloroethyl, β(dibutylmethylsilyl)ethyl, p-toluenesulfonylethyl,4-nitrobenzylsulfonylethyl, allyl, cinnamyl,1-(trimethylsilylmethyl)prop-1-en-3-yl and like moieties. Preferredcarboxy-protecting groups are allyl, benzyl and t-butyl. Furtherexamples of these groups are found in E. Haslam, "Protective Groups inOrganic Chemistry", J. G. W. McOmie, Ed., Plenum Press, New York, N.Y.,1973, Chapter 5, and T. W. Greene, "Protective Groups in OrganicSynthesis", John Wiley and Sons, New York, N.Y., 1981, Chapter 5.

Preferred compounds used in the claimed method are those compounds offormula I where:

R⁰ is hydrogen, hydroxy, C₁ -C₆ alkyl, C₁ -C₆ alkoxy, hydroxy(C₁ -C₆alkyl), --X⁰ --O--C(O)--C₁ -C₄ alkyl, --O--(X¹)_(i) --X², --C(O)--X³ or--O--R³ ;

R¹ is hydrogen, hydroxy, C₁ -C₆ alkoxy, sulfhydryl, sulfamyl, --SO₂--Cl, amino, di(C₁ -C₄ alkylsulfonyl)amino, --C(O)--X³, --N--C(O)--R² or--O--R³ ;

X⁰ is a bond or divalent(C₁ -C₆ alkyl);

X¹ is an amino acid;

X² is hydrogen or an amino protecting group;

i is 1 or 2;

X³ is C₁ -C₆ alkyl;

R² is hydroxy(C₁ -C₄ alkyl);

R³ is C₂ -C₆ alkenyl, --CH₂ --R^(3a), --C(O)--R^(3b), --C(S)--R^(3c),--C(CH₃)₂ C(O)NH₂ or a group of the formula: ##STR3## R^(3a) is phenyl,p-fluorophenyl, pyridyl, piperidinyl, piperazinyl or morpholinyl;

R^(3b) is piperidinyl, piperazinyl, morpholinyl, N--(C₁ -C₄alkoxycarbonyl)piperidinyl, N-(trifluoromethyl)piperidinyl, halo(C₁ -C₄alkoxy) or di(C₁ -C₄ alkyl)amino;

R^(3c) is di(C₁ -C₄ alkyl)amino;

R^(3d) is oxygen or hydroximino;

R^(3e) is hydrogen, nitro or trifluoromethyl;

X is a bond;

R⁴ is hydrogen, hydroxy, amino, ═O, C₂ -C₆ alkanoyloxy, ═N--R⁵,--OSi(CH₃)₂ or R⁴ and R⁶ combine to form a bond;

R⁵ is hydroxy, amino, di(C₁ -C₄ alkyl)amino, C₁ -C₄ alkoxy,pyridylmethoxy, N-(methyl)piperazinyl or --O--CH₂ --C(O)--R^(5a) ;

R⁶ is hydrogen, chloro, bromo, methyl or ═O;

R⁷ is hydrogen or methyl;

R⁸ is hydroxy, chloro, methoxy, 4-methylpiperazinyl or --N(R⁹)--R¹⁰ ;

R⁹ is hydrogen;

R¹⁰ is --CH₂ --R^(10a) ; and

R^(10a) is pyridyl; or a pharmaceutically acceptable salt thereof.

Of these compounds, more preferred are those compounds of formula Iwhere:

R⁰ is hydrogen, hydroxy, C₁ -C₆ alkoxy, --O--(X¹)_(i) --X², --X⁰--O--C(O)--C₁ -C₄ alkyl or --O--R³ ;

R¹ is hydrogen, hydroxy, C₁ -C₆ alkoxy or --O--R³ ;

X⁰ is a bond;

X¹ is an amino acid;

X² is hydrogen or an amino protecting group;

i is 1 or 2;

R³ is C₂ -C₆ alkenyl, --CH₂ --R^(3a) or --C(O)--R^(3b) ;

R^(3a) is p-fluorophenyl or pyridyl;

R^(3b) is piperidinyl;

R⁴ is hydrogen, hydroxy, ═O or ═N--R⁵ ;

R⁵ is hydroxy, dimethylamino or N-(methyl)piperazinyl;

R⁶ is hydrogen, bromo or ═O;

R⁷ is methyl; and

R⁸ is methoxy; or a pharmaceutically acceptable salt thereof.

Of these compounds, even more preferred are those compounds of formula Iwhere:

R⁰ is hydrogen, hydroxy, C₁ -C₄ alkoxy, --O--(X¹)_(i) --X²,--O--C(O)methyl or --O--R³ ;

R¹ is hydrogen, hydroxy, C₁ -C₄ alkoxy or --O--R³ ;

X¹ is glycine, alanine or valine;

X² is hydrogen, t-butoxycarbonyl or benzyloxycarbonyl;

R⁴ is ═O or ═N--R⁵ ;

R⁵ is hydroxy;

R⁶ is hydrogen; or a pharmaceutically acceptable salt thereof.

The compounds of formula I may be prepared according to procedures knownin the art. For example, the following Reaction Schemes may be used,alone or in combination to provide the desired compounds. Once areaction is complete, the intermediate compound may be isolated byprocedures well-known in the art, for example, the compound may becrystallized and then collected by filtration, or the reaction solventmay be removed by extraction, evaporation or decantation. Theintermediate compound may be further purified, if desired, by commontechniques such as crystallization or chromatography over solid supportssuch as silica gel or alumina, before carrying out the next step of thereaction scheme.

The compounds of formula I where R⁴ is ═O or ═N--R may be preparedaccording to the procedures shown below in Reaction Scheme I. ##STR4##where Reactions I.4A and 4B represent alternative reactions that followReaction I.3.

Reaction scheme I is accomplished by carrying out reactions 1-4 issequential order. Reaction I.1 is carried out by oxidizing a compound offormula IA, for example, by reaction with chromium trioxide in an aceticacid/water mixture, to provide the corresponding ketone. The chromiumtrioxide is generally employed in an amount ranging from equimolarproportions to about a 4 molar excess relative to the compound offormula IA, preferably in about a 2-4 molar excess. The aceticacid/water mixture is generally a 10:1 to a 2:1 mixture of acetic acidto water, preferably about 4:1. The reaction is generally substantiallycomplete after about 1 to 10 hours when conducted at a temperature offrom about 23° C. to about 60° C. The reaction is preferably conductedat a temperature of from about 23° C. to about 30° C. for about 5 to 10hours.

In Reaction I.2, the ketone obtained from Reaction I.1 is reacted withbromine in a suitable solvent such as diethyl ether, tetrahydrofuran ordimethoxyethane, to provide a mixture of bromoketones which are thenseparated using standard separation techniques such as chromatography.These isomerically pure bromoketones are then used to prepare variousisomerically pure compounds of formula I. The bromine is generallyemployed in an amount ranging from about equimolar proportions to abouta 2 molar excess relative to the ketone reactant, preferably in about a1-1.5 molar excess. Solvent choice is not critical so long as thesolvent employed is inert to the ongoing reaction and the reactants aresufficiently solubilized to effect the desired reaction. The reaction isgenerally substantially complete after about 1 to 3 hours when conductedat a temperature of from about 23° C. to about 30° C. The reaction ispreferably conducted at room temperature for about 1 to 1.5 hours.

Alternatively, the ketone obtained from Reaction I.1 is reacted with asilylating agent in the presence of a base in a suitable solvent such asmethylene chloride, diethyl ether or tetrahydrofuran to provide thecorresponding silylated enol ether. Preferred bases include 2,6-lutidineand collidine. A preferred silylating agent is t-butyldimethyl-silyltrifluoromethanesulfonate. The silylating agent is generally employed inan amount ranging from about equimolar proportions to about a 2 molarexcess relative to the ketone reactant, preferably in about a 1-1.5molar excess. Solvent choice is not critical so long as the solventemployed is inert to the ongoing reaction and the reactants aresufficiently solubilized to effect the desired reaction. The reaction isgenerally substantially complete after about 30 minutes to 2 hours whenconducted at a temperature of from about 0° C. to about 50° C. Thereaction is preferably conducted at a temperature of from about 10° C.to about 25° C. for about 30 minutes to about 1 hour.

The silylated enol ether is then reacted with bromine substantially asdescribed above with the exception that the reaction is carried out inthe presence of acetic acid. Typical solvents suitable for use in thisreaction include any organic solvent such as methylene chloride, diethylether or tetrahydrofuran. Solvent choice is not critical so long as thesolvent employed is inert to the ongoing reaction and the reactants aresufficiently solubilized to effect the desired reaction.

In Reaction I.3, the bromoketone is reduced, for example by reactionwith zinc dust and sodium acetate in glacial acetic acid, to provide thecorresponding ketones. The zinc is generally employed in an amountranging from about equimolar proportions to about a 4 molar excessrelative to the ketone reactant, preferably in about a 1.5-3 molarexcess. The sodium acetate is generally employed in an amount rangingfrom about 0.6 molar equivalents to about 1.2 molar equivalents relativeto the ketone reactant. The reaction is generally substantially completeafter about 1 to 10 hours when conducted at a temperature of from about60° C. to the reflux temperature of the mixture. The reaction ispreferably conducted at reflux temperature for about 1 to 2 hours.

Alternatively, hydroxylamine hydrochloride is reacted with sodiumacetate in a suitable solvent such as ethanol. The sodium acetate isgenerally employed in an amount ranging from about 1.1 molar equivalentsto about a 50 molar excess relative to the hydroxylamine. The reactionis generally substantially complete after about 1 to 72 hours whenconducted at a temperature of from about 25° C. to about 80° C. Thereaction is preferably conducted at a temperature in the range of fromabout 25° C. to about 30° C. for about 5 to 24 hours.

In Reaction I.4A, the ketone obtained from Reaction I.3 is reacted withhydroxylamine hydrochloride in a mixture of methanol, water and aceticacid to provide the desired oxime compound. The hydroxylaminehydrochloride is generally employed in an amount ranging from aboutequimolar proportions to about a 4 molar excess relative to the ketonereactant, preferably in about a 1.3-3 molar excess. The ratio ofmethanol to water to acetic acid is generally 10-20:1:0.1, preferably15:1:0.1. The reaction is generally substantially complete after about 1hour to about 2 days when conducted at a temperature of from about 40°C. to the reflux temperature of the mixture. The reaction is preferablyconducted at reflux temperature for about 1 to 6 hours.

In Reaction I.4B, the ketone obtained from Reaction I.3 is reacted withan hydrazine hydrochloride such as 1-amino-4-methylpiperazine,1,1-dimethylhydrazine or hydrazine in the presence of a base in an inertsolvent at a temperature of from about 25° C. to 80° C. for 2 to 24hours. Typical bases include sodium acetate, potassium hydroxide,triethylamine and the like. Suitable solvents include ethanol,isopropanol and dimethylformamide. Solvent choice is not critical solong as the solvent employed is inert to the ongoing reaction and thereactants are sufficiently solubilized to effect the desired reaction.

The phenyl moiety of the compounds of formula I prepared above may besubstituted according to Reaction Scheme II, as follows: ##STR5## whereR⁰ and R¹ are independently hydrogen or --C(O)CH₃ ; and R⁰ " and R¹ 'are independently hydrogen or hydroxy.

In Reaction II.1, the compound of formula I where R⁰ and R¹ are eachhydrogen is subjected to a Friedel-Crafts acylation by reacting thecompound of formula I with an acid halide, in the presence of a catalystin an inert solvent such as carbon disulfide. The acid halide isgenerally employed in an amount ranging from about equimolar proportionsto about a 1.5 molar excess relative to the compound of formula I,preferably in about a 1.1-1.3 molar excess. Preferred acid halidesinclude acetyl chloride, acetyl bromide or the like. Preferred catalystsinclude aluminum trichloride, aluminum tribromide or the like. Solventchoice is not critical so long as the solvent employed is inert to theongoing reaction and the reactants are sufficiently solubilized toeffect the desired reaction. The reaction is generally substantiallycomplete after about 1 to 10 hours when conducted at a temperature offrom about 50° C. to the reflux temperature of the mixture. The reactionis preferably conducted at reflux temperature for about 1 to 2 hours.

In Reaction II.2, the acylated compound of formula I obtained fromReaction II.1 is oxidized to provide the corresponding phenol in a twostep reaction. First, the acyl moiety is reacted with a peracid in thepresence of an acid catalyst in an inert solvent such as dimethoxyethaneto provide the corresponding ester with is then reacted with sodiumbicarbonate in an alcohol/water mixture to provide the desired phenol.

The peracid is generally employed in an amount ranging from aboutequimolar proportions to about a 2 molar excess relative to the acylmoiety, preferably in about a 1-1.3 molar excess. The amount of catalysttypically employed is in the range of 0.005-0.04 equivalents relative tothe acyl moiety. A preferred peracid is metachloro-peroxybenzoic acid. Apreferred catalyst is p-toluenesulfonic acid. Solvent choice is notcritical so long as the solvent employed is inert to the ongoingreaction and the reactants are sufficiently solubilized to effect thedesired reaction. The reaction is generally substantially complete afterabout 1 to 10 hours when conducted at a temperature of from about 50° C.to the reflux temperature of the mixture. The reaction is preferablyconducted at reflux temperature for about 1 to 3 hours.

The resultant ester is typically refluxed with a base in amethanol/water mixture for about 1 to 7 hours to provide the desiredphenol compound. Preferred bases include sodium bicarbonate, sodiumcarbonate, sodium hydroxide or potassium hydroxide or the like. The baseis generally employed in an excess, for example from about a 1 molarexcess to about a 6 molar excess relative to the ester moiety,preferably in about a 2-5 molar excess.

The phenol compounds obtained from Reaction Scheme II may be used toprepare various substituted compounds of formula I, as described below.

For example, the hydroxy moiety may be alkylated by reacting the phenolcompound with a suitable alkylating agent in the presence of a base inan inert solvent. Examples of bases include triethylamine, diisopropylethylamine, sodium hydride and potassium carbonate. Typical solventsinclude methylene chloride, tetrahydrofuran, dimethylformamide and thelike. Solvent choice is not critical so long as the solvent employed isinert to the ongoing reaction and the reactants are sufficientlysolubilized to effect the desired reaction. Suitable alkylating agentsinclude iodomethane, allyl iodide, p-fluorophenyl bromide,3-bromomethyl-pyridine and 2-fluorobenzophenone and the like. Thereaction is generally substantially complete after about 1 to 20 hourswhen conducted at a temperature of from about 0° C. to 170° C. Thereaction is preferably conducted at a temperature of from about 25° C.to about 80° C. for about 4 to 16 hours.

Alternatively, the hydroxy moiety may be alkylated by reacting thephenol with an alcohol in the presence of triphenylphosphine and asuitable activating agent in an inert solvent, such as tetrahydrofuranor ethylene glycol dimethyl ether. Examples of suitable activatingagents include diethyl azodicarboxylate, dimethyl azodicarboxylate,diisopropyl azodicarboxylate and the like. Examples of alcohols include3-pyridyl carbinol, N-t-butoxycarbonyl-3-piperidinemethanol and thelike. The reaction is generally substantially complete after about 0.5to 2 hours when conducted at a temperature of from about 0° C. to 85° C.The reaction is preferably conducted at a temperature of from about 25°C. to about 70° C. for about 30 minutes to 1 hour.

The hydroxy moiety may be converted to an ester or a carbonate byreacting the phenol with an acylating agent in the presence of a base inan inert solvent, such as methylene chloride, tetrahydrofuran ordimethylformamide. Typical bases include triethylamine, diisopropylethylamine, sodium hydride and the like. Typical acylating agentsinclude N-(t-butoxycarbonyl)-4-chlorocarbonyl piperdine,2,2,2-trichloroethyl chloroformate,N-(t-butoxycarbonyl)-hydroxybenzotriazole amino esters. The reaction isgenerally substantially complete after about 1 to 20 hours whenconducted at a temperature of from about 0° C. to 60° C. The reaction ispreferably conducted at a temperature of from about 10° C. to about 25°C. for about 1 to 5 hours.

The hydroxy moiety may be converted to the corresponding aniline in athree step reaction. First, the phenol is reacted with a suitablysubstituted amide such as 2-methyl-2-bromo-propanamide in the presenceof a base such as sodium hydride or triethylamine in an inert solvent,such as dioxane or tetrahydrofuran at a temperature of 25° C. to 100° C.to provide the corresponding amido-ether. This amido-ether is thenreacted with sodium hydride in an inert solvent such asdimethylformamide, 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidone or amixture thereof at temperatures ranging from 25° C. to 145° C. toprovide the rearranged amido-alcohol. Finally, the amido-alcohol isreacted with an acid, such as hydrochloric acid in dioxane at 50° C. to100° C. to provide the desired aniline.

The aniline may be converted to the corresponding sulfonamide byreacting the aniline with a sulfonyl chloride such as methanesulfonylchloride or isopropylsulfonyl chloride in the presence of a base, suchas triethylamine, diisopropyl ethylamine or sodium hydride at atemperature of from about 0° C. to 50° C. in an inert solvent, such asmethylene chloride, tetrahydrofuran or dimethylformamide.

The hydroxy moiety may be converted to a thiophenol in a three stepreaction. First the phenol is reacted with a thio-carbamoyl (for exampledimethylthiocarbamoyl chloride) in the presence of a base in an suitablesolvent, such as water or dimethylformamide at a temperature rangingfrom 25° C. to 50° C. for 1 to 3 hours to provide the oxo-thiocarbamate.Typical bases include potassium hydroxide, triethylamine and the like.The oxo-thiocarbamate is converted to the correspondingthio-oxocarbamate compound by isolating and heating the neat solid toits melting point. Finally, the thio-oxocarbamate is reacted with abase, such as potassium hydroxide or sodium hydroxide in an alcoholicsolvent, such as methanol or ethanol at a temperature of 20° C. to 80°C. for 20 minutes to 1 hour to provide the corresponding thiophenol.

The thiophenol may be converted to the corresponding sulfonamides byreacting the thiophenol with an oxidizing agent (for example, potassiumnitrate) in an inert solvent such as acetonitrile, followed by theaddition of a chlorinating agent (for example, sulfuryl chloride) attemperatures ranging from 0° C. to 25° C. to provide a mixture ofsulfonyl chlorides which are separable using standard chromatographictechniques. These sulfonyl chlorides may be converted to the desiredsulfonamides by reaction with an appropriately substituted amine such asammonium hydroxide, methylamine, isopropylamine or benzylamine at atemperature of from about 0° C. to 40° C. in an inert solvent suchtetrahydrofuran.

The hydroxy moiety may be converted to the corresponding amino esters byreacting the phenol with an amino protected amino acid in the presenceof a coupling reagent and a catalyst in an inert solvent such as diethylether, tetrahydrofuran or methylene chloride. Preferred amino protectinggroups include t-butoxycarbonyl or benzyloxycarbonyl. The amino reactantis generally employed in equimolar proportions to a slight excess (1.3equivalents) relative to the phenol reactant in the presence of anequimolar quantity to a slight excess (1.5 equivalents) of the couplingreagent. Typical coupling agents include dicyclohexylcarbodiimide (DCC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide,benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate(BOP), N,N'-diethylcarbodiimide, carbonyldiimidazole,bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP--Cl) orN-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) and the like.Preferred coupling agents include DCC and BOP. Typical catalysts includeDMAP and 4-pyrrolopyridine. The reaction is substantially complete in 1to 10 hours when carried out at a temperature of from about -30° C. toabout 35° C., preferably from about 0° C. to about 25° C.

The starting materials used in the procedures detailed above may beobtained commercially or prepared according to procedures known in theart. For example, methyl O-methylpodocarpate having the followingstereochemistry may be obtained from Aldrich Chemical Company: ##STR6##

In addition, the compound(s) of formula IA, below may be preparedsubstantially in accordance with the procedure detailed in Ohta andOhmuri, Chem. Pharm. Bull. (Tokyo), vol 5, page 91 (1957). The isomericmix of compounds may be separated using standard separation techniques.Preferably, these isomers are obtained using the bromination methodologydescribed above in Reaction Scheme I.

The compound(s) of formula IA may also be used to prepare other isomersusing the procedure detailed in Pelletier et al., Tetr. Lett. page 4179(1971). For example, heating the compound(s) of formula IA in a highboiling point solvent such as triethylene glycol dimethylether(triglyme) results in a compound of formula IB as follows: ##STR7##

The resultant mixture of isomers is then separated using standardprocedures such as recrystallization or column chromatography or may besubjected to the bromination methodology described above in ReactionScheme I.

The following Preparations and Examples further illustrate specificaspects of the present invention. It is to be understood, however, thatthese examples are included for illustrative purposes only and are notintended to limit the scope of the invention in any respect and shouldnot be so construed. ##STR8##

To a solution of NaOMe (prepared in situ from 2.6 g of sodium in 400 mlof anhydrous MeOH (0.108 mol), under N₂), was added 15.0 g (0.035 mol)of 70% abietic acid. After stirring the mixture for 10 minutes, 14.0 ml(0.22 mol) of iodomethane was added and the mixture was refluxed for 24hours, cooled and concentrated in vacuo to provide a residue. Thisresidue was dissolved in 500 ml of EtOAc, washed sequentially with 500ml of a saturated NaHCO₃ solution and a saturated sodium chloridesolution (NaCl), dried over Na₂ SO₄, filtered and concentrated in vacuo.The crude material was purified using flash chromatography (eluent of 2%EtOAc in hexanes). Yield: 10.0 g of a dark yellow oil (90.4%).IR(CHCl₃): 2952, 1718 and 1251 cm⁻¹. ¹ H NMR (300MHz, CDCl₃): δ5.78 (s,1H); 5.38 (brs, 1H); 3.66 (s, 3H); 2.17-2.30 (m, 3H); 1.68-2.16 (m, 8H);1.50-1.65 (m, 2H); 1.26 (s, 3H); 1.24 (m, 2H); 1.02 (d, J=2.6Hz, 3H);1.00 (d, J=2.6Hz, 3H) and 0.83 (s, 3H). MS(FD): m/e 316(M+).

Elemental Analysis for C₂₁ H₃₂ O₂ : Calcd: C, 79.70; H, 10.19;Found: C,79.49; H, 9.94. ##STR9##

To a mixture of 5.0 g (15.8 mmol) of the compound in Preparation 1 in100 ml of acetic anhydride, was added 2.5 g (22.5 mmol) of selenium (IV)oxide, under N₂. The reaction mixture was warmed to 70° C., stirred for16 hours, cooled, filtered and then diluted to 500 ml with CH₂ Cl₂. Theresulting layers were separated and the organic layer was washed with500 ml of NaCl, dried over Na₂ SO₄, filtered and then concentrated invacuo to provide a dark yellow solid. This solid was purified usingflash chromatography (eluent of 5% EtOAc in hexanes) to provide twomajor fractions.

The first fraction was concentrated to provide 537 mg of an oil. Thisoil was hydrogenated with 135 mg of 5% Pd/C in 25 ml of MeOH (8 hours,room temperature, 6.0 psi). The reaction mixture was filtered and thefiltrate concentrated in vacuo. The crude material was purified usingflash chromatography (eluent of 2% EtOAc in hexanes) to provide thecompound of Preparation 3 (400 mg of a clear oil (75%) m.p. 50° C.). Thesecond fraction was concentrated in vacuo to provide the compound.Yield: 2.8 g of a light yellow solid (47%). m.p. 165-167° C. IR (KBr):2956, 1722 and 1251 cm⁻¹. ¹ H NMR (300MHz, CDCl₃): δ7.23 (m, 2H); 7.04(d, J=1.8Hz, 1H); 5.90 (m, 1H); 3.64 (s, 3H); 2.86 (m, 1H); 2.60 (dd,J=1.5,11.0Hz, 1H); 2.31 (d, J=12.1Hz, 1H); 2.08 (s, 3H); 2.07 (m, 1H);1.60-1.80 (m, 6H); 1.26 (s, 3H); 1.24 (s, 3H); 1.22 (s, 3H) and 1.19 (s,3H). MS(FD): m/e 372(M+).

Elemental Analysis for C₂₃ H₃₂ O₄ : Calcd: C, 74.16; H, 8.66; Found: C,74.44; H, 8.71. ##STR10##

To a mixture of 23.6 g (0.063 mmol) of the compound of Preparation 2 in1500 ml of MeOH, was added 5.8 g of 10% Pd/C and 5.8 g (0.030 mmol) ofp-toluenesulfonic acid monohydrate. The reaction mixture was reacted for16 hours at room temperature, 60 psi, filtered and then concentrated invacuo to provide a residue. This residue was dissolved in 700 ml ofEtOAc, washed sequentially with 700 ml of saturated NaHCO₃ and NaClsolutions, dried over Na₂ SO₄, filtered and then concentrated in vacuo.Yield: 19.3 g (97.5%) of an oil. IR (CHCl₃): 2955, 1718 and 1254 cm⁻¹. ¹H NMR (300MHz, CDCl₃): δ7.16 (d, J=8Hz, 1H); 7.00 (d, J=8Hz, 1H); 6.88(s, 1H); 3.66 (s, 3H); 2.80-2.90 (m, 3H); 2.23-2.32 (m, 2H); 1.35-1.90(m, 7H); 1.28 (s, 3H); 1.24 (s, 3H) and 1.21 (s, 6H). MS(FD): m/e314(M+).

Elemental Analysis for C₂₁ H₃₀ O₂ : Calcd: C, 80.21; H, 9.62 Found: C,80.34; H, 9.73 ##STR11##

A mixture of 475 mg (1.5 mmol) of the compound of Preparation 3, 425 mg(3.19 mmol) of anhydrous aluminum chloride in 15 ml of toluene wasstirred at room temperature for 2 hours, under N₂. The reaction mixturewas partitioned between toluene and 1N HCl. The resultant layers wereseparated and the organic layer was washed with brine, dried over Na₂SO₄, filtered and concentrated in vacuo to provide an oil. This oil waspurified using flash chromatography (SiO₂, eluent of 2% EtOAc inhexanes) to provide an oil which was crystallized from MeOH ¹ H NMR(300MHz, CDCl₃): δ7.00-7.30 (m, 4H); 3.30 (s, 1.5H); 3.28 (s, 1.5H);2.90 (m, 2H); 2.30 (m, 2H); 2.00 (m, 1H); 1.40-1.80 (m, 6H); 1.30 (s,1.5H); 1.22 (s, 3H) and 1.10 (s, 1.5H). ##STR12##

A solution of 285 mg (2.8 mmol) of chromium trioxide in 4 ml of glacialAcOH and 1 ml of H₂ O was added dropwise to a solution of 275 mg (1mmol) of the compound of Preparation 4 in 5 ml of glacial AcOH. Thereaction mixture was stirred at room temperature for 2 hours and thenpartitioned between EtOAc and brine (twice). The combined organic layerswere dried over Na₂ SO₄, filtered and then concentrated in vacuo toprovide a yellow oil which was purified using flash chromatography(SiO₂, eluent of 5% EtOAc in hexanes). Yield: 50 mg of a bright yellowsolid (17%). m.p. 121-123° C. IR(CHCl₃): 3019, 2954, 1727, 1688 and 1248cm⁻¹. ¹ H NMR (300MHz, CDCl₃): δ8.14 (d, J=8Hz, 1H); 7.70 (7, J=7Hz,1H); 7.47 (m, 2H); 3.73 (s, 3H); 3.39 (s, 1H); 2.64 (d, J=12Hz, 1H);2.01-2.11 (m, 1H); 1.40-1.80 (m, 4H); 1.29 (s, 3H) and 0.69 (s, 3H).MS(FD): m/e 300(M+).

Elemental Analysis for C₁₈ H₂₀ O₄ : Calcd: C, 71.98; H, 6.71; Found: C,72.10; H, 6.66. ##STR13##

The compound was isolated from the reaction mixture of Example 1. Yield:136 mg of an oil (47.5%). ¹ H NMR (300MHz, CDCl₃): δ8.01 (m, 1H); 7.55(m, 1H); 7.30 (m, 2H); 3.30 (s, 1.5H); 3.28 (s, 1.5H); 3.10 (dd,J=4,12Hz, 0.5H): 2.70 (m, 1.5H); 2.40 (m, 2H); 1.40-1.90 (m, 5H); 1.30(s, 1.5H); 1.28 (s, 1.5H); 1.23 (s, 1.5H); 0.65 (s, 1.5H). ##STR14##

A solution of 0.9 ml (17 mmol) of bromine in 30 ml of anhydrous Et₂ Owas added to a solution of 3.8 g (13.3 mmol) of the compound of Example2 in 200 ml of anhydrous Et₂ O, dropwise. The reaction mixture wasstirred at room temperature for 1 hour and then washed sequentially withH₂ O, a saturated NaHCO₃ solution and 19% sodium thiosulfate, dried overNa₂ SO₄, filtered and concentrated in vacuo to provide a residue whichwas purified using flash chromatography (eluent of 3:2 CH₂ Cl₂/hexanes). Yield: 1.2 g of yellowish oil (25%). ¹ H NMR (300MHz, CDCl₃):δ8.00 (dd, J=2,5Hz, 1H); 7.60 (dt, J=2,5Hz, 1H); 7.40 (m, 2H); 4.60 (s,1H); 3.78 (s, 3H); 3.25 (s, 1H) 2.50 (d, J=7Hz, 1H); 1.60-1.90 (m, 5H);1.60 (s 3H) ; 0.57 (s, 3H). ##STR15##

A mixture of 1.2 g (3.3 mmol) of the compound of Example 3, 450 mg (6.9mmol) of zinc dust, 225 mg (2.7 mmol) of NaOAc and 50 ml of glacial AcOHwas refluxed for 1 hour, under N₂. After cooling, the reaction mixturewas filtered and the filtrate was partitioned between Et₂ O and brine.The resultant layers were separated and the organic layer was dried overNa₂ SO₄, filtered and then concentrated in vacuo. The crude material waspurified using flash chromatography (SiO₂, eluent of 10% Et₂ O inhexanes). Yield: 897 mg of a pale yellow oil (93%). IR(CHCl₃): 3018,1721, 1675 and 1257 cm⁻¹. ¹ H NMR (300MHz, CDCl₃): δ8.03 (m, 1H); 7.55(m, 1H); 7.30 (m, 2H); 3.69 (s, 3H); 3.13 (dd, J=7,19Hz, 1H); 2.76 (dd,J=3,7Hz, 1H); 2.50 (d, J=3Hz, 1H); 2.45 (7, J=3Hz, 1H); 1.88 (m, 1H);1.58 (m, 4H); 1.33 (s, 3H) and 0.69 (s, 3H). MS(FD): m/e 286(M+).

Elemental Analysis for C₁₈ H₂₂ O₃ : Calcd: C, 75.50; H, 7.74; Found: C,75.75; H, 7.89. ##STR16##

A mixture containing of the compound of Example 4, hydroxylaminehydrochloride, NaHCO₃, glacial AcOH, in a H₂ O/MeOH mixture was refluxedwith a Dean-Stark trap for 5 hours. The reaction mixture wasconcentrated in vacuo to provide a residue which was partitioned betweenH₂ O and CH₂ Cl₂ and the organic layer was dried over Na₂ SO₄, filteredand concentrated in vacuo. The crude material was purified using flashchromatography. Yield: 98%. IR(CHCl₃): 3583, 2952 and 1720 cm⁻¹. ¹ H NMR(300MHz, CDCl₃): δ7.86 (d, J=8.8Hz, 1H); 7.20-7.38 (m, 4H); 3.70 (s,3H); 3.10 (dd, J=8.1,19.8Hz, 1H); 2.53-2.64 (m, 2H); 2.47 (d, 11.4Hz,1H); 1.70-1.90 (m, 1H); 1.40-1.60 (m, 4H); 1.19 (s, 3H) and 0.56 (s,3H). MS(FD): m/e 302(M+).

Elemental Analysis for C₁₈ H₂₃ NO₃ : Calcd: C, 71.73; H, 7.69; N, 4.65;Found: C, 71.79; H, 7.78, N, 4.44. ##STR17##

To a solution of 295 mg (0.98 mmol) of the compound of Example 5 in 5 mlof anhydrous DMF, was added 60 mg (1.50 mmol) of 60% NaH on mineral oil,under N₂, followed by the addition of 0.18 ml (1.90 mmol) of methylbromoacetate was added by syringe. The reaction mixture was stirred for1 hour and then cautiously quenched by the dropwise addition of brineunder N₂. The reaction mixture was partitioned between brine and Et₂ O,the resulting layers were separated and the organic layer was dried overNa₂ SO₄, filtered and the concentrated in vacuo to provide an oil. Thisoil was purified using flash chromatography (SiO₂, eluent of 20% Et₂ Oin hexanes). Yield: 175 mg of a clear oil (48%). IR(CHCl₃): 2953, 1737and 1725 cm⁻¹. ¹ H NMR (300MHz, CDCl₃): δ7.87 (d, J=8Hz, 1H); 7.29 (m,2H); 7.19 (s, J=8Hz, 1H); 4.75 (s, 2H); 3.76 (s, 3H); 3.69 (s, 3H); 3.17(dd, J=8,20Hz, 1H); 2.35-2.57 (m, 3H); 1.73-1.85 (m, 1H); 1.44-1.61 (m,4H); 1.20 (s, 3H) and 0.56 (s, 3H). MS(FD): m/e 373 (M+).

Elemental Analysis for C₂₁ H₂₇ NO₅ : Calcd: C, 67.54; H, 7.29; N, 3.75;Found: C, 67.84; H, 7.58; N, 3.89. ##STR18##

A mixture of 56.7 mg (0.152 mmol) of the compound of Example 6, 0.2 ml(0.2 mmol) of 1N NaOH and 5 ml of MeOH was stirred at room temperaturefor 4 days and then diluted to 30 ml with brine and extracted withEtOAc. The layers were separated. The aqueous layer was acidified with5N HCl and extracted with EtOAc and the combined organic layers weredried over Na₂ SO₄, filtered and then concentrated in vacuo. Yield: 45.7mg of an amorphous tan resin (84%). IR(CHCl₃): 3030, 2951 and 1720 cm⁻¹.¹ H NMR (300MHz, CDCl₃): δ12.75 (brs, 1H); 7.76 (d, J=8Hz, 1H); 7.40 (m,2H); 7.21 (m, 1H); 4.66 (s, 2H); 3.62 (s, 3H); 3.05 (dd, J=8,20Hz, 1H);2.47 (m, 3H); 1.60-1.80 (m, 1H); 1.20-1.59 (m, 4H); 1.10 (s, 3H) and0.42 (s, 3H). MS(FD): m/e 359 (M+)

Elemental Analysis for C₂₀ H₂₅ NO₅ ·0.25H₂ O: Calcd: C, 66.05; H, 7.01;N, 3.85; Found: C, 65.91; H, 7.35; N, 3.61. ##STR19##

A mixture of 111.8 mg (0.31 mmol) of the compound of Example 7, 0.31 ml(0.31 mmol) of 1N NaOH and 10 ml of anhydrous CH₃ CN was sonicated for30 minutes and then concentrated in vacuo to provide a residue. Thisresidue was repetitively concentrated from fresh Et₂ O. Yield: 116 mg ofan amorphous solid (98%). IR(KBr): 3429, 2948, 1725 and 1611 cm¹. ¹ HNMR (300MHz, CDCl₃): δ7.75 (d, J=7.4Hz, 1H); 7.32 (m, 2H); 7.20 (m, 1H);4.20 (s, 2H); 3.63 (s, 3H); 3.00 (dd, J=7.7,19.1.Hz, 1H); 2.35-2.50 (m,3H); 1.60-1.80 (m, 1H); 1.40-1.60 (m, 4H); 1.10 (s, 3H) and 0.43 (s,3H). MS(FD): m/e 285 (M+--C₂ H₂ O₃ Na). ##STR20##

A mixture of 4.0 g (14.7 mmol) of the compound of Preparation 4 and 1.2ml (16.9 mmol) of acetyl chloride in 60 ml of carbon disulfide to asuspension of 2.6 mg (19.5 mmol) of anhydrous aluminum chloride in 100ml of carbon disulfide, via dropping funnel. The reaction mixture wasrefluxed for 1 hour and then the carbon disulfide was removed bydownward distillation. The resultant mixture was cautiously quenched bythe addition of 100 ml of 0.2N HCl. The desired compound was extractedusing 100 ml of CH₂ Cl₂, dried over Na₂ SO₄, filtered and thenconcentrated in vacuo to provide a dark red oil. This oil was purifiedusing flash chromatography (SiO₂, eluent of 20% Et₂ O in hexanes).Yield: 1.7 g of an oil (87% based on recovered starting material). ¹ HNMR (300MHz, CDCl₃): δ7.90 (d, J=4Hz, 1H); 7.75 (d, J=4Hz, 0.5H); 7.63(d, J=4Hz, 0.5H); 7.37 (d, J=6Hz, 0.5H); 7.10 (d, J=6Hz, 0.5H); 3.70 (s,1.5H); 3.68 (s, 1.5H); 2.92 (m, 2H); 2.60 (s, 3H); 2.00-2.50 (m, 3H);1.40-1.98 (m, 6H); 1.29 (s, 1.5H); 1.26 (s, 1.5H); 1.24 (s, 1.5H) and1.10 (s, 1.5H). ##STR21##

A mixture of 1.7 g (5.4 mmol) of the compound of Example 9A, 1.9 g (5.5mmol) of 50% 3-chloroperoxybenzoic acid, 18 mg (0.095 mmol) of p-toluenesulfonic acid monohydrate in 25 ml of 1,2-dimethyoxyethane was refluxedfor 3 hours, under N₂. After cooling, the reaction mixture was dilutedwith Et₂ O and washed sequentially with 10% potassium iodide, 10% sodiumthiosulfate, a saturated NaHCO₃ solution and brine, dried over Na₂ SO₄,filtered and then concentrated to provide a resin. This resin wasdissolved in 25 ml of MeOH and 10 ml of H₂ O containing 1.6 g (19.0mmol) of NaHCO₃. The resultant mixture was refluxed for 1.5 hours,cooled, filtered and concentrated in vacuo to provide a residue. Thisresidue was partitioned between H₂ O and Et₂ O. The resulting layerswere separated and the organic layer was washed sequentially with 1N HCland brine, dried over Na₂ SO₄, filtered and concentrated in vacuo Yield:1.55 g (99%). ¹ H NMR (300MHz, CDCl₃): δ6.85 (m, 1H); 6.70 (d, J=6Hz,1H); 6.55 (dd, J=6Hz, 1H); 3.63 (s, 3H); 2.80 (m, 2H); 1.90-2.30 (m,3H); 1.40-1.88 (m, 6H); 1.25 (s, 1.5H); 1.20 (s, 1.5H); 1.17 (s, 1.5H)and 1.02 (s, 1.5H). ##STR22##

To a suspension of 1.55 g (5.4 mmol) of the compound of Example 9B and275 mg (6.87 mmol) of 60% NaH on mineral oil in 50 ml of anhydrous DMF,was added 0.5 ml (7.50 mmol) of idodomethane, under N₂. The reactionmixture was stirred for 1 hour and then cautiously quenched by thedropwise addition of brine. The reaction mixture was partitioned betweenEt₂ O and brine. The resultant layers were separated and the organiclayer was dried over Na₂ SO₄, filtered and concentrated in vacuo toprovide a residue which was purified using flash chromatography (SiO₂,eluent of 20% Et₂ O in hexanes). Yield: 1.3 g of a clear light yellowoil (88.5% based on recovered starting material). ¹ H NMR (300MHz,CDCl₃): δ7.00 (d, J=6Hz, 1H); 6.82 (m, 1H); 6.70 (m, 1H); 3.80 (s, 3H);3.70 (s, 3H); 2.80 (m, 2H); 2.00-2.40 (m, 3H); 1.40-1.90 (m, 6H); 1.25(s, 1.5H); 1.20 (s, 3H); 1.10 (s, 1.5H). Note: The reaction mixture alsocontained 200 mg of the compound of Example 9A. ##STR23##

The compound was prepared substantially in accordance with the proceduredetailed in Example 1, using 1.3 g (4.3 mmol) of the compound of Example9C. The crude material was purified using flash chromatography (SiO₂,eluent of 20% Et₂ O in hexanes). Yield: 820 mg of an oil (60.5%). ¹ HNMR (300MHz, CDCl₃): δ8.00 (m, 1H); 6.80 (m, 2H); 3.87 (s 1.5H); 3.85(s, 1.5H); 3.63 (s, 1.5H); 3.61 (s, 1.5H); 3.01 (dd, J=5, 12Hz, 0.5H);2.70 (m, 1.5H); 2.40 (m, 2H); 1.40-1.95 (m, 5H); 1.32 (s, 1.5H); 1.30(s, 1.5H); 1.22 (s, 1.5H); 0.65 (s, 1.5H). ##STR24##

The compound was isolated from the reaction mixture in Example 9D.Yield: 35.4 mg of an oil. ##STR25##

The compound was isolated from the reaction mixture in Example 9D.Yield: 150 mg of an oil (10.6%). ¹ H NMR (300MHz, CDCl₃): δ8.17 (d,J=6Hz, 1H); 6.95 (m, 1H); 6.80 (m, 1H); 3.90 (s, 3H); 3.70 (s, 3H); 3.36(s, 0.5H); 2.50 (m, 1.5H); 1.80-2.10 (m, 1H); 1.42-1.80 (m, 4H); 1.40(s, 1.5H); 1.20 (s, 1.5H); 1.17 (s, 1.5H); 0.65 (s, 1.5H). ##STR26##

The compound was obtained by separating the compounds (52 mg) of Example9E by radial chromatography (eluent of 15% Et₂ O in hexanes). Yield:20.5 mg (1.1%) (overall yield). ¹ H NMR (300MHz, CDCl₃): δ7.52 (d,J=2Hz, 1H); 7.21 (d, J=6Hz, 1H); 7.10 (dd, J=2.6Hz, 1H); 3.87 (s, 3H);3.63 (s, 3H); 3.05 (dd, J=4,12Hz, 1H); 2.72 (m, 1H); 2.20-2.50 (m, 2H);1.80 (m, 2H); 1.40-1.60 (m, 3H); 1.30 (s, 3H) and 0.70 (s, 3H). MS(FD):m/e 316 (M+). ##STR27##

The compound was prepared substantially in accordance with the proceduredetailed in Example 3, using 975 mg (3.08 mmol) of the compound ofExample 9D. The crude material was purified using flash chromatography(SiO₂, eluent of 15% of Et₂ O in hexanes). Yield: 532.6 mg (44%). ¹ HNMR (300MHz, CDCl₃): δ8.03 (d, J=6Hz, 1H); 6.81 (m, 2H); 4.50 (s, 1H);3.90 (s, 3H); 3.75 (s, 3H); 3.21 (s, 1H); 2.40 (m, 1H); 1.60-2.00 (m,5H); 1.58 (s, 3H) and 0.60 (s, 3H). ##STR28##

The compound was prepared substantially in accordance with the procedureof Example 4, using 532.0 mg (1.35 mmol) of the compound of Example 9H.Yield: 280 mg of a pale yellow solid (66%). m.p. 117-119° C. IR(CHCl₃):2942, 1721, 1667 and 1596 cm⁻¹. ¹ H NMR (300MHz, CDCl₃): δ8.04 (d,J=9Hz, 1H); 6.83 (s, 1H); 6.80 (d, J=3Hz, 1H); 3.90 (s, 3H); 3.67 (s,3H); 3.02 (dd, J=7,19Hz, 1H); 2.72 (dd, J=2,7Hz, 1H); 2.40 (m, 2H);1.80-1.90 (m, 1H); 1.45-1.65 (m, 4H); 1.31 (s, 3H); 0.70 (s, 3H).MS(FD): m/e 316 (M+).

Elemental Analysis for C₁₉ H₂₄ O₄ : Calcd: C, 72.13; H, 7.65; Found: C,72.43; H, 7.67. ##STR29##

The compound was prepared substantially in accordance with the proceduredetailed in Example 5, using 465 mg (1.17 mmol) of the compound ofExample 9I. Yield: 79%. IR(CHCl₃): 3500, 2951 and 1719 cm⁻¹. ¹ H NMR(300MHz, CDCl₃): δ0.81 (d, J=7Hz, 1H); 6.82 (d, J=2Hz, 1H); 6.78 (dd,J=2,7Hz, 1H); 3.83 (s, 3H); 3.70 (s, 3H); 3.07 (dd, J=6,12Hz, 1H); 2.60(m, 2H); 2.40 (m, 1H), 1.80 (m, 1H); 1.40-1.60 (m, 4H); 1.20 (s, 3H) and0.60 (s, 3H). MS(FD): m/e 331 (M+).

Elemental Analysis for C₁₉ H₂₅ NO₄ : Calcd: C, 68.86; H, 7.60; N, 4.23;Found: C, 68.56; H, 7.44; N, 4.25. ##STR30##

The compound was prepared substantially in accordance with the proceduredetailed in Example 62, using the compound of Example 98G. Yield: 90%. ¹H NMR (300MHz, CDCl₃): δ7.40 (d, J=2Hz, 1H); 7.20 (d, J=6Hz, 1H); 6.93(dd, J=2,6Hz, 1H); 3.82 (s, 3H); 3.70 (s, 3H); 3.10 (dd, J=5,13Hz, 1H);2.50-2.63 (m, 2H); 2.1 (d, J=8Hz, 1H); 2.30 (m, 1H); 1.20-1.40 (m, 4H);1.20 (s, 3H) and 0.60 (s, 3H). MS(FD): m/e 331 (M+).

As noted above, the compounds of the present invention are useful forinhibiting an envelope virus that undergoes hemagglutinin-mediatedfusion with a host cell. Thus, the claimed compounds may be used totreat or prevent a viral infection where the virus is an envelope virusthat undergoes hemagglutinin-mediated fusion which comprisesadministering to an virus-infected cell, a cell susceptible to infectionor a mammal in need thereof an effective amount of a compound of formulaI or a pharmaceutically acceptable salt thereof. The claimed compoundsmay also be used to inhibit viral replication in an envelope virus thatundergoes hemagglutinin-mediated fusion which comprises administering toa virus-infected cell, a cell susceptible to infection or a mammal inneed thereof, an effective amount of a compound of formula I or apharmaceutically acceptable salt thereof.

The term "effective amount" as used herein, means an amount of acompound of the present invention which is capable of inhibiting thehemagglutinin mediated fusion of the virus with the host cell. Theinhibition contemplated by the present method includes both therapeuticand prophylactic treatment, as appropriate. The specific dose ofcompound administered according to this invention to obtain therapeuticand/or prophylactic effects will, of course, be determined by theparticular circumstances surrounding the case, including, for example,the compound administered, the route of administration, the conditionbeing treated and the individual being treated. A typical daily dose(administered in single or divided doses) will contain a dosage level offrom about 0.01 mg/kg to about 50 mg/kg of body weight of an activecompound of this invention. Preferred daily doses generally will be fromabout 0.05 mg/kg to about 20 mg/kg and ideally from about 0.1 mg/kg toabout 10 mg/kg.

The compounds can be administered by a variety of routes including oral,rectal, transdermal, subcutaneous, intravenous, intramuscular andintranasal. The compounds of the present invention are preferablyformulated prior to administration. Therefore, another embodiment of thepresent invention is a pharmaceutical formulation comprising aneffective amount of a compound of formula I or a pharmaceuticallyacceptable salt thereof and a pharmaceutically acceptable carrier,diluent or excipient therefor.

The active ingredient in such formulations comprises from 0.1% to 99.9%by weight of the formulation. By "pharmaceutically acceptable" it ismeant that the carrier, diluent or excipient is compatible with theother ingredients of the formulation and not deleterious to therecipient thereof.

The present pharmaceutical formulations are prepared by known proceduresusing known and readily available ingredients. In making thecompositions of the present invention, the active ingredient willusually be admixed with a carrier, or diluted by a carrier, or enclosedwithin a carrier which may be in the form of a capsule, sachet, paper orother container. When the carrier serves as a diluent, it may be asolid, semi-solid or liquid material which acts as a vehicle, excipientor medium for the active ingredient. Thus, the compositions can be inthe form of tablets, pills, powders, lozenges, sachets, cachets,elixirs, suspensions, emulsions, solutions, syrups, aerosols, (as asolid or in a liquid medium), ointments containing, for example, up to10% by weight of the active compound, soft and hard gelatin capsules,suppositories, sterile injectable solutions, sterile packaged powdersand the like.

The following experiments were carried out to demonstrate the ability ofthe compounds of the present invention to inhibit influenza.

In Vitro CPE/XTT Assay

MDCK cells were dispersed in a microtiter plate (96 wells) at 10,000cells per well with Medium 199 containing Earl's balanced salt solution(EBSS), 1% fetal bovine serum (FBS), penicillin (100 units/ml) andstreptomycin (100 μg/ml). After standing overnight at 37° C. in a carbondioxide (CO₂) incubator, the MDCK cells were infected with ˜0.1 moi(mutiplicity of infection) of influenza virus (i.e. A/Kawasaki/89 orB/Hong Kong and B/Great Lakes) at 0.03 moi. After allowing the virus toadsorb to the cells for 1-2 hours, medium containing serial dilutions ofdrug or medium alone was added to the wells. The resultant mixtures wereincubated for 2-3 days (until extensive cpe was apparent in medium alonewells). The antiviral effect of a test compound was assessed byperforming the following XTT assay.

A fresh solution (0.4 mg/ml) of XTT[2,3-bis(methoxy-4-nitro-5-sulfophenyl)-2H-tetraazolium-5-carboxanilide,inner salt, sodium salt] in warm medium without FBS was prepared. Foreach 5 ml of the XTT solution, 25 μl of 5mM PMS (phenazine methosulfate)in phosphate buffer saline was added. After withdrawing the culturedsupernatant, 100 μl of the freshly prepared XTT/PMS mixture was added toeach of the microtiter wells. The wells were then incubated at 37° C.(under CO₂) for 3-4 hours or until color change is prominent. Theabsorbance at 450 nm (ref. 650 nm) was read in a spectrophotometer. Theconcentration of test compound required to cause 50% cytotoxic effect(TC₅₀) relative to a control with no drug and no virus present and whichinhibits the development of virus cytopathic effect (cpe) by 50% (IC₅₀)or 90% (IC₉₀) was determined from the linear portion of each doseresponse curve.

Using this CPE/XTT assay, the IC₅₀ of the compounds of formula I wasdetermined to be less than 0.1 μg/ml for influenza A/Kawasaki/89 andgreater than 100 μg/ml for influenza B/Lee.

Plague Reduction Assay

Susceptible MDCK cells were grown in 6 well tissue culture treatedcluster plates at 1×10⁶ cells/well in Minimum 199 with 1 percent fetalbovine serum, penicillin (100 units/ml) and streptomycin (100 μg/ml).After overnight incubation at 37° C., the growth medium was removed and0.2 ml/well of an appropriate dilution of virus was added. Afteradsorption for 1-2 hour at room temperature, the infected cell sheet wasoverlaid with equal parts of 1.5% sterile agarose solution and a twofoldconcentration of medium 199 (with 2% fetal bovine serum, 100 units/ml ofpenicillin and 100 μg/ml streptomycin) containing varying concentrationsof compounds.

The compounds were dissolved in DMSO at a concentration of 20 mg/ml andan aliquot was diluted to the desired concentration in DMSO and thenadded to the agar medium mixture. The plates were incubated in a CO₂incubator at 37° C. until the DMSO control wells contained plaques ofoptimal size. Then, a solution containing 10 percent formalin and 2percent sodium acetate was added to each well to inactivate the virusand fix the cell sheet to the plastic surface. The fixed cell sheetswere stained with 0.5 percent crystal violet and the plaques werecounted. Results from duplicate wells at each concentration wereaveraged and compared with DMSO control wells. The inhibition of plaqueformation by 50 or 90 percent (IC₅₀ or IC₉₀) was calculated from thelinear region of the inhibition concentration curve using the method ofReed and Muench, Am. J. Hyg., vol. 27, pages 493-497 (1958).

Using this plaque reduction assay, the IC₅₀ of the compounds of formulaI was determined to be in the range of 0.01-5.9 μg/ml for influenzaA/Kawasaki and was determined to be in the range of 3.3 μg/ml to 19μg/ml for influenza B/Lee.

What is claimed is:
 1. A compound of the formula ##STR31## wherein: R⁰and R¹ are independently hydrogen, hydroxy, C₁ -C₆ alkyl, C₁ -C₆ alkoxy,hydroxy(C₁ -C₆ alkyl), sulfhydryl, sulfamyl, --SO₂ --Cl,--S--C(O)--N(CH₃)₂, amino, C₁ -C₄ alkylamino, di(C₁ -C₄ alkyl)amino, C₁-C₄ alkylsulfonylamino, di(C₁ -C₄ alkylsulfonyl)amino, --X⁰--O--C(O)--C₁ -C₄ alkyl, --O--(X¹)_(i), --C(O)--X³, --N--C(O)--R² or--O--R³ ;X⁰ is a bond or divalent(C₁ -C₆ alkyl); X¹ is an amino acidester of glycine, alanine or valine; i is 1, 2 or 3; X³ is C₁ -C₆ alkyl,C₁ -C₆ alkoxy, halo(C₁ -C₆ alkyl), hydroxy(C₁ -C₆ alkyl) or phenyl; R²is C₁ -C₄ alkyl, C₁ -C₄ alkoxy, halo(C₁ -C₄ alkyl), hydroxy(C₁ -C₄alkyl), phenyl, p-methoxy-phenyl, p-fluoro-phenyl, naphthyl, orcyclohexyl; R³ is C₂ -C₆ alkenyl, --CH₂ --R^(3a), --C(O)--R^(3b),--C(S)--R^(3c), --C(CH₃)₂ C(O)NH₂, phenyl or a group of the formula##STR32## R^(3a) is phenyl, p-fluorophenyl, cyclohexyl, cyclopentyl,cyclopropyl or naphthyl; R^(3b) is benzyloxy, C₁ -C₆ alkoxy, halo(C₁ -C₄alkoxy), amino, C₁ -C₄ alkylamino or di(C₁ -C₄ alkyl)amino; R^(3c) isamino, C₁ -C₄ alkylamino or di(C₁ -C₄ alkyl)amino; R^(3d) is oxygen,hydroximino, hydrazino or ═CHZ; Z is hydrogen, C₁ -C₄ alkyl, halogen,di(C₁ -C₄ alkyl)amino, C₁ -C₄ alkoxycarbonyl, carbamoyl(C₁ -C₄ alkyl),N--(C₁ -C₄ alkyl)carbamoyl or N,N-di(C₁ -C₄ alkyl)carbamoyl; R^(3e) ishydrogen, nitro or trifluoromethyl; X is a bond or --(CH₂)--; R⁴ is═N--R⁵ ; R⁵ is hydroxy, amino, C₁ -C₄ alkylamino, di(C₁ -C₄ alkyl)amino,C₁ -C₄ alkoxy, benzyloxy, or --O--CH₂ --C(O)--R^(5a) ; R^(5a) is hydroxyor C₁ -C₄ alkoxy; R⁶ is hydrogen, halo, C₁ -C₄ alkyl or ═O; R⁷ ishydrogen or C₁ -C₄ alkyl; R⁸ is hydroxy, halo, or C₁ -C₆ alkoxy; or apharmaceutically acceptable salt thereof provided that R⁰ is notisopropyl.
 2. The compound of claim 1 whereinR⁰ is hydrogen, hydroxy, C₁-C₆ alkyl, C₁ -C₆ alkoxy, hydroxy(C₁ -C₆ alkyl), --X⁰ --O--C(O)--C₁ -C₄alkyl, --O--(X¹)_(i), --C(O)--X³ or --O--R³ ; R¹ is hydrogen, hydroxy,C₁ -C₆ alkoxy, sulfhydryl, sulfamyl, --SO₂ --Cl, amino, di(C₁ -C₄alkylsulfonyl)amino --C(O)--X³, --N--C(O)--R² or --O--R³ ; X⁰ is a bondor divalent (C₁ -C₆ alkyl); X¹ is an amino acid ester of glycine,alanine or valine; i is 1 or 2; X³ is C₁ -C₆ alkyl; R² is hydroxy(C₁ -C₄alkyl); R³ is C₂ -C₆ alkenyl, --CH₂ --R^(3a), --C(O)--R^(3b),--C(S)--R^(3c), --C(CH₃)₂ C(O)NH₂ or a group of the formula: ##STR33##R^(3a) is phenyl, or p-fluorophenyl; R^(3b) is halo(C₁ -C₄ alkoxy) ordi(C₁ -C₄ alkyl)amino; R^(3c) is di(C₁ -C₄ alkyl)amino; R^(3d) is oxygenor hydroximino; R^(3e) is hydrogen, nitro or trifluoromethyl; X is abond; R⁴ is ═N--R⁵ ; R⁵ is hydroxy, amino, di(C₁ -C₄ alkyl)amino, C₁ -C₄alkoxy, or --O--CH₂ --C(O)--R^(5a) ; R⁶ is hydrogen, chloro, bromo,methyl or ═O; R⁷ is hydrogen or methyl; R⁸ is hydroxy, chloro, ormethoxy; or a pharmaceutically acceptable salt thereof.
 3. The compoundof claim 2 whereinR⁰ is hydrogen, hydroxy, C₁ -C₆ alkoxy, --O--(X¹)_(i),--X⁰ --O--C(O)--C₁ -C₄ alkyl or --O--R³ ; R¹ is hydrogen, hydroxy, C₁-C₆ alkoxy or --O--R³ ; X⁰ is a bond; X¹ is an amino acid ester ofglycine, alanine or valine; i is 1 or 2; R³ is C₂ -C₆ alkenyl, or --CH₂--R^(3a) ; R^(3a) is p-fluorophenyl; R⁴ is ═N--R⁵ ; R⁵ is hydroxy, ordimethylamino; R⁶ is hydrogen, bromo or ═O; R⁷ is methyl; and R⁸ ismethoxy; or a pharmaceutically acceptable salt thereof.
 4. The compoundof claim 3 whereinR⁰ is hydrogen, hydroxy, C₁ -C₄ alkoxy, --O--(X¹)_(i),--O--C(O)methyl or --O--R³ ; R¹ is hydrogen, hydroxy, C₁ -C₄ alkoxy or--O--R³ ; X¹ is an amino acid ester of glycine, alanine or valine; R⁴ is═N--R⁵ ; R⁵ is hydroxy; R⁶ is hydrogen; or a pharmaceutically acceptablesalt thereof.
 5. The compound of claim 1 wherein R⁴ is ═N--OH.
 6. Thecompound of claim 2 wherein R⁴ is ═N--OH.
 7. The compound of claim 3wherein R⁴ is ═N--OH.